Large-scale synthesis of "Cpep" RNA monomers and their application in automated RNA synthesis

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2'-protecting group for large-scale syntheses. Unlike 2'-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2'-deprotection. The syntheses of 2'-Cpep protected nucleosides (A, C, G, and U) has been accomplished on a 0.5 Kg scale. The 2'-Cpep monomers were transformed into 3'-O-phosphoramidites for conventional automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and initial automated solid-phase synthesis are demonstrated.     

Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.     

RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry

The chemical synthesis of RNA oligonucleotides is a valuable resource for biological research. A new approach for RNA synthesis that is now as reliable and efficient as DNA synthesis methods is described in this report. A 5'-O-silyl ether is used in conjunction with acid-labile orthoester protecting groups on the 2'-hydroxyls. RNA synthesis proceeds efficiently on commercial synthesizers in high yields. Analysis by anion-exchange HPLC shows that the quality and yields of RNA synthesized with this chemistry are unprecedented. Furthermore, this chemistry enables analysis and purification of stable 2'-O-protected RNA. This property serves to minimize possibilities for degradation of the RNA. In addition, it now possible to analyze troublesome sequences, which, when fully 2'-O-deprotected, do not easily resolve into one major conformation due to strong secondary structure. When ready for use, the RNA is easily 2'-O-deprotected in mild-acidic aqueous buffers in 30 min. This new RNA chemistry has enabled the routine high-quality synthesis of RNA oligonucleotides up to 50 bases in length regardless of sequence or secondary structure.     

Synthesis of isotopically labeled D-[1'-13C]ribonucleoside phosphoramidites

The preparation of fully protected labeled diisopropylamino-beta-cyanoethyl-[1'-13C]ribonucleoside phosphoramidites with regioisomeric purity is described. We demonstrated in this paper that a regioselective 2'-O-silylation, through a 3',5'-O-di-tert-butylsilanediyl protection, has been applied for the synthesis of [1'-13C]ribonucleoside phosphoramidite units. This method allowed us to obtain only the desired 2'-O-silyl-3'-O-phosphoramidites avoiding the undesired 3'-O-silyl-2'-O-phosphoramidite nucleosides isolated by standard procedures. This is a suitable procedure to RNA precursors with respect to the isotope-containing precursors.     

A simple, rapid and effective method for total RNA extraction from Lentinula edodes

A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms.     

Protective groups in the chemical synthesis of oligoribonucleotides

The methods of chemical oligoribonucleotide synthesis and the protective groups used are reviewed. The latest data on the protection of 2′-OH functions of nucleotide derivatives used as monomers for the RNA synthesis are comprehensively discussed.     

siRNA as a tool for streamlining functional genomic studies

RNA interference (RNAi) has the potential to accelerate greatly the pace of discovery biology. The active RNAi intermediate is the small interfering RNA (siRNA), a discrete nucleic acid duplex that can be generated by several methods and used to directly silence gene expression. The choice of methods employed depends largely on the research or therapeutic objective. In most cases, rational design offers several advantages over random design, including greater predictability of function, higher silencing potency and longer duration of suppression. Of the production methods, chemical synthesis provides the fastest production capability, the highest purity and the easiest scalability for high-throughput strategies. Effective coupling of several methods gives the greatest potential for the application of RNAi across functional genomic and target validation studies. Furthermore, the coupling of RNAi with cellular profiling technologies will provide opportunities to streamline drug discovery significantly.     

Facile Protection-Free One-Pot Chemical Synthesis of Nucleoside-5′-Tetraphosphates

A new, straightforward, reliable, and convenient protection-free one-pot method for the synthesis of 2′-deoxynucleoside-5′-tetraphosphate and ribonucleoside-5′-tetraphosphate is reported. The present synthetic strategy involves the monophosphorylation of a nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and subsequent hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide nucleoside-5′-tetraphosphate in moderate yield with high purity. A plausible mechanism is proposed to account for the formation of product.     

Oligonucleotide analysis by gel capillary electrophoresis

Several million oligonucleotides are synthesized each year for a broad variety of molecular biology applications. Steady improvements in the synthesis chemistry efficiency and the automated DNA synthesizers have made production of oligonucleotides routine and reliable. Many applications, such as PCR and sequencing, are often successful when the primers have not been rigorously purified. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary for the dozens of oligonucleotides produced each day by a DNA synthesis laboratory. Traditional methods of analysis have been HPLC and polyacrylamide slab tel electrophoresis (PAGE). Gel capillary electrophoresis is a new option, combining the advantages of the HPLC and PAGE, with unprecedented resolution and speed.     

On the synthesis of orexin A: a novel one-step procedure to obtain peptides with two intramolecular disulphide bonds.

An efficient strategy for the synthesis of orexin A, a recently discovered neuropeptide with two intramolecular disulphide bonds, was developed. Four different methods for the synthesis of peptides containing two disulphide bonds were compared and optimized with respect to reaction time, purity of the crude peptide and yield of the purified peptide. A new one-step cyclization method in solution is presented for fast, easy and high yield synthesis of orexin A, based on iodine oxidation in acetic acid/water and S-acetamidomethyl (S-Acm) and S-trityl (S-Trt) for side-chain protection of cysteine. Disulphide formation without selective side-chain protection leads to the formation of different mono- and bicyclic configurations of orexin A. These data stress the requirement of selective cysteine side-chain protection in the synthesis of orexin A.     

Postsynthetic on column RNA labeling via Stille coupling

Stille Coupling is a versatile C-C bond forming reaction with high functional group tolerance under mild conditions. Our on column synthesis concept for RNA modification is based on the incorporation of iodo substituted nucleotide precursors to RNA during automated standard solid phase synthesis via TBDMS-, TC-, and ACE- protecting group strategies. Subsequently, the RNA, still bound on solid support, is ready for orthogonal postsynthetic functionalization via Stille cross-couplings utilizing the advantages of solid phase synthesis. Several monomer test reactions were employed with 2-iodo adenosine and 5-iodo uridine and organostannanes as coupling partners under different conditions, changing the catalyst/ligand system, temperature, and reaction time as well as conventional heating and microwave irradiation. Finally, Stille cross-couplings under optimized conditions were transferred to fully protected 5-mer and 12-mer RNA oligonucleotides on-column. Deprotection and cleavage from solid support resulted in site-specifically labeled oligonucleotides. Derivatizations via Stille cross-couplings were performed initially with vinyltributylstannane as well as later with 2-furanyl-, 2-thiophene-, and benzothiophene-2-tributylstannanes yielding fluorescently functionalized RNA.     

New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure.     

Optimal synthesis of protein purification processes

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.     

Turnover of polyadenylated messenger RNA in fission yeast. Evidence for the control of protein synthesis at the translational level.

Polyadenylated RNA was isolated from fission yeast (Schizosaccharomyces pombe) total RNA using oligo(dT)-cellulose, and was studied as a model for messenger RNA. The half-life of poly adenylated RNA was measured by two independent methods. (a) The rate of labelling of polyadenylated RNA during incubation of cells with [5-3H]uridine was measured. A half-life of 40-45 min was found by comparing the experimental data with theoretical curves calculated for labelling of RNAs with various half-lives. The influence of precursor-pool specific activity on RNA labelling kinetics is considered. (b) Cells were labelled with [5-3H]uridine then further RNA synthesis was inhibited by addition of 8-hydroxyquinoline. The rate of loos of radioactivity from polyadenylated RNA indicated a half-life of 50 min. The half-life found by these two methods is about one-third of the cell doubling time, and is much longer than previous estimates by indirect methods of yeast messenger RNA half-life. Both experimental methods provided evidence for the existence of tas a half-life of 40-50 min; a much smaller population is probably turning over more rapidly. After inhibition of RNA synthesis by 8-hydroxyquinoline, the rate of total protein synthesis declined much more rapidly than the polyadenylated RNA content of the cells. However, 60 min after inhibition of RNA synthesis there was a small rise in the rate of portein synthesis. These data are interpreted as evidence for mechanisms controlling protein synthesis which operate at the level of messenger RNA translation.     

Rapid solid-phase synthesis of a calmodulin-binding peptide using controlled microwave irradiation

A rapid and efficient microwave-assisted solid-phase synthesis method for the preparation of a nonapeptide using conventional Fmoc/Bu(t) orthogonal protection strategy is described. In this protocol, the coupling steps are performed within 5 min at 60 degrees C and the Fmoc-deprotection steps are completed within 3 min at 60 degrees C using a dedicated single-mode microwave peptide synthesizer utilizing temperature-controlled conditions. It is demonstrated that the model nonapeptide (containing the calmodulin-binding octapeptide sequence) is synthesized in a shorter time (approximately 3.5 h) and with high purity (>95%) under microwave irradiation conditions in comparison with a reference peptide that is obtained by standard methods at room temperature (within 11 h).     

Mechanism of ribonucleic acid chain initiation. 1. A non-steady-state study of ribonucleic acid synthesis without enzyme turnover

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.